Establishment of drug screening model targeting PRDM1 gene promoter / 重庆医学

ABSTRACT

Objective To construct a double luciferase reporter gene vector with PRDM1 gene promoter as target,and establish drug screening cell model in vitro,hope to find small molecule compounds in Chinese herbal medicine library by this model.Methods Total DNA was extracted from 293T cells and it was used for amplifying the fragment contained PRDM1 gene promoter (267 bp/1 257 bp) by PCR.The amplified product was inserted into pGL3-Basic vector.The PCR product and pGL3-PRDM1 vector were verified by sequencing and alignment.The pGL3-PRDM1 and pRL-TK vector were co-transfected into engineer cells.The activity of PRDM1 gene promoter could be assayed by measuring luciferase.The method was optimized by changing ratio of two vectors.Results The highest transfection efficiency and luciferase activity were found in ratio of n(pGL-PRDM1) ∶ n(pRL-TK)=2 ∶1,and with the recombinant luciferase report gene vector contained the length of 1 257 bp amplified fragment transfecting into U266 cells.Moreover,the inductor (5-Aza-CdR) of PRDM1 gene was used for authenticating the method(P＜0.01),the fluoresscence in tensity and promoter activity of PRDM1 gene were enhanced in a dose dependent manner with 5-Aza-CdR.The activity of the promoter of PRDM1 gene was significantly decreased from the concentration of 5 μmol/L of Artemisinid(P＜0.05).The total glucosides of paeoniflorin promoted the promoter activity of PRDM1 gene at a low concentration,and inhibited the promoter activity of PRDM1 gene at a high concentration.Artesunate has no effect on cell proliferation.The effect of total glucosides of paeony on cell proliferation was more complicated.Conclusion A drug selection model targeting PRDM1 gene promoter has been successfully established,and artesunate has been screened to inhibit the promoter activity of PRDM1 gene.